Peter J. Hurlin

Mad3 and Mad4: novel Max-interacting transcriptional repressors that suppress c-myc dependent transformation and are expressed during neural and epidermal differentiation.

The basic helix‐loop‐helix‐leucine zipper (bHLHZip) protein Max associates with members of the Myc family, as well as with the related proteins Mad (Mad1) and Mxi1. Whereas both Myc:Max and Mad:Max heterodimers bind related E‐box sequences, Myc:Max activates transcription and promotes proliferation while Mad:Max represses transcription and suppresses Myc dependent transformation. Here we report the identification and characterization of two novel Mad1‐ and Mxi1‐related proteins, Mad3 and Mad4. Mad3 and Mad4 interact with both Max and mSin3 and repress transcription from a promoter containing CACGTG binding sites. Using a rat embryo fibroblast transformation assay, we show that both Mad3 and Mad4 inhibit c‐Myc dependent cell transformation. An examination of the expression patterns of all mad genes during murine embryogenesis reveals that mad1, mad3 and mad4 are expressed primarily in growth‐arrested differentiating cells. mxi1 is also expressed in differentiating cells, but is co‐expressed with either c‐myc, N‐myc, or both in proliferating cells of the developing central nervous system and the epidermis. In the developing central nervous system and epidermis, downregulation of myc genes occurs concomitant with upregulation of mad family genes. These expression patterns, together with the demonstrated ability of Mad family proteins to interfere with the proliferation promoting activities of Myc, suggest that the regulated expression of Myc and Mad family proteins function in a concerted fashion to regulate cell growth in differentiating tissues.

Targeted disruption of the MYC antagonist MAD1 inhibits cell cycle exit during granulocyte differentiation

The switch from transcriptionally activating MYC–MAX to transcriptionally repressing MAD1–MAX protein heterodimers has been correlated with the initiation of terminal differentiation in many cell types. To investigate the function of MAD1–MAX dimers during differentiation, we disrupted the Mad1 gene by homologous recombination in mice. Analysis of hematopoietic differentiation in homozygous mutant animals revealed that cell cycle exit of granulocytic precursors was inhibited following the colony‐forming cell stage, resulting in increased proliferation and delayed terminal differentiation of low proliferative potential cluster‐forming cells. Surprisingly, the numbers of terminally differentiated bone marrow and peripheral blood granulocytes were essentially unchanged in Mad1 null mice. This imbalance between the frequencies of precursor and mature granulocytes was correlated with a compensatory decrease in granulocytic cluster‐forming cell survival under apoptosis‐inducing conditions. In addition, recovery of the peripheral granulocyte compartment following bone marrow ablation was significantly enhanced in Mad1 knockout mice. Two Mad1‐related genes, Mxi1 and Mad3, were found to be expressed ectopically in adult spleen, indicating that functional redundancy and cross‐regulation between MAD family members may allow for apparently normal differentiation in the absence of MAD1. These findings demonstrate that MAD1 regulates cell cycle withdrawal during a late stage of granulocyte differentiation, and suggest that the relative levels of MYC versus MAD1 mediate a balance between cell proliferation and terminal differentiation.

Sequential expression of the MAD family of transcriptional repressors during differentiation and development

Members of the Myc proto-oncogene family encode transcription factors that function in multiple aspects of cell behavior, including proliferation, differentiation, transformation and apoptosis. Recent studies have shown that MYC activities are modulated by a network of nuclear bHLH-Zip proteins. The MAX protein is at the center of this network in that it associates with MYC as well as with the family of MAD proteins: MAD1, MXI1, MAD3 and MAD4. Whereas MYC–MAX complexes activate transcription, MAD–MAX complexes repress transcription through identical E-box binding sites. MAD proteins therefore act as antagonists of MYC. Here we report the expression patterns of the Mad gene family in the adult and developing mouse. High level of Mad gene expression in the adult is limited to tissues that display constant renewal of differentiated cell populations. In embryos, Mad transcripts are widely distributed with expression peaking during organogenesis at the onset of differentiation. A detailed analysis of their pattern of expression during chrondrocyte and neuronal differentiation in vivo, and during neuronal differentiation of P19 cells in vitro, shows that Mad family genes are sequentially induced. Mad3 transcripts and proteins are detected in proliferating cells prior to differentiation. Mxi1 and Mad4 transcripts are most abundant in cells that have further advanced along the differentiation pathway, whereas Mad1 is primarily expressed late in differentiation. Taken together, our data suggest that the different members of the MAD protein family exert their functions at distinct steps during the transition between proliferation and differentiation.

Tbx3 impinges on the p53 pathway to suppress apoptosis, facilitate cell transformation and block myogenic differentiation

Tbx3 is a member of the T-box family of transcription factors. Mutations in Tbx3 cause ulnar-mammary syndrome, an autosomal dominant disorder characterized by upper limb defects, apocrine-gland defects including mammary hypoplasia, and tooth, hair and genital defects. In cell culture, Tbx3 and its close relative Tbx2 are capable of immortalizing mouse embryo fibroblasts. We show that expression of Tbx3 together with Myc or oncogenic Ras (H-RasVal17) leads to efficient transformation of mouse embryo fibroblasts. Oncogene cooperation by Tbx3 correlates with an ability of Tbx3 to suppress the induction of p19ARF and p53 that is typically caused by overexpression Myc and Ras, and to protect against Myc-induced apoptosis. Whereas Tbx3 is capable of interfering with apoptosis caused by excessive Myc levels, a Tbx3 mutant lacking its C-terminal repression domain shows no anti-apoptotic activity and fails to repress levels of p19ARF or p53. Consistent with an ability to suppress p53 pathway function, we find that Tbx3, but not a Tbx3 C-terminal mutant, efficiently blocks myogenic differentiation of C2C12 myoblasts. Our results support the idea that deregulation and/or excessive levels of Tbx3 may have oncogenic potential in vivo.

Deletion of Mnt leads to disrupted cell cycle control and tumorigenesis

Mnt is a Max‐interacting transcriptional repressor that has been hypothesized to function as a Myc antagonist. To investigate Mnt function we deleted the Mnt gene in mice. Since mice lacking Mnt were born severely runted and typically died within several days of birth, mouse embryo fibroblasts (MEFs) derived from these mice and conditional Mnt knockout mice were used in this study. In the absence of Mnt, MEFs prematurely entered the S phase of the cell cycle and proliferated more rapidly than Mnt+/+ MEFs. Defective cell cycle control in the absence of Mnt is linked to upregulation of Cdk4 and cyclin E and the Cdk4 gene appears to be a direct target of Mnt–Myc antagonism. Like MEFs that overexpress Myc, Mnt−/− MEFs were prone to apoptosis, efficiently escaped senescence and could be transformed with oncogenic Ras alone. Consistent with Mnt functioning as a tumor suppressor, conditional inactivation of Mnt in breast epithelium led to adenocarinomas. These results demonstrate a unique negative regulatory role for Mnt in governing key Myc functions associated with cell proliferation and tumorigenesis.

The interplay between Mad and Myc in proliferation and differentiation

Members of the Myc family of transcription factors are key regulators of cell proliferation, and excessive levels of Myc lead to tumor formation. Mad family proteins are related to Myc, but they antagonize the oncogenic activity of Myc in cell-culture assays. Here, we examine current models of Mad function and the relationship between Mad and Myc in cell proliferation, differentiation and tumorigenesis.

Mnt–Max to Myc–Max complex switching regulates cell cycle entry

The c-Myc oncoprotein is strongly induced during the G0 to S-phase transition and is an important regulator of cell cycle entry. In contrast to c-Myc, the putative Myc antagonist Mnt is maintained at a constant level during cell cycle entry. Mnt and Myc require interaction with Max for specific DNA binding at E-box sites, but have opposing transcriptional activities. Here, we show that c-Myc induction during cell cycle entry leads to a transient decrease in Mnt–Max complexes and a transient switch in the ratio of Mnt–Max to c-Myc–Max on shared target genes. Mnt overexpression suppressed cell cycle entry and cell proliferation, suggesting that the ratio of Mnt–Max to c-Myc–Max is critical for cell cycle entry. Furthermore, simultaneous Cre-Lox mediated deletion of Mnt and c-Myc in mouse embryo fibroblasts rescued the cell cycle entry and proliferative block caused by c-Myc ablation alone. These results demonstrate that Mnt-Myc antagonism plays a fundamental role in regulating cell cycle entry and proliferation.

Functions of Myc:Max in the Control of Cell Proliferation and Tumorigenesis

Deregulation and elevated expression of members of the Myc family of bHLHZip transcription factors are observed in a high percentage of tumors. This close association with human cancers has led to a tremendous effort to define their biological and biochemical activities. Although Myc family proteins have the capacity to elicit a wide range of cell behaviors, their principal function appears to be to drive cells into the cell cycle and to keep them there. However, forced expression of Myc profoundly sensitizes normal cells to apoptosis. Therefore, tumor formation caused by deregulated Myc expression requires cooperating events that disrupt pathways that mediate apoptosis. Myc-dependent tumor formation may also be impeded by a set of related bHLHZip proteins with the demonstrated potential to act as Myc antagonists in cell culture experiments. In this review, we examine the complex activities of Myc family proteins and how their actions might be regulated in the context of a network of bHLHZip proteins.

The Max Transcription Factor Network: Involvement of Mad in Differentiation and an Approach to Identification of Target Genes

The small bHLHZip protein, Max, was originally identified through its interaction with Myc family proteins and appears to be an obligate partner for Myc function. Max has now been found to interact with at least two other proteins, Mad and Mxi1. These also belong to the bHLHZip class but are otherwise unrelated to Myc. Mad has been shown to abrogate the positive transcriptional activity of Myc and to inhibit Myc in co-transformation assays. This suggests that Mad may antagonize Myc function. Mad is rapidly induced upon differentiation, a time when Myc is frequently down-regulated. We show here evidence for Mad expression upon differentiation of myeloblasts, monoblasts, and keratinocytes. Mad:Max complexes are detected during differentiation and appear to replace the Myc:Max complexes present in proliferating cell populations. Since these complexes appear to form even in the presence of Myc, there may exist mechanisms that act to inhibit Myc:Max, or to promote Mad:Max, complex formation. We speculate that Max complex switching causes a change in the transcriptional activity of groups of target genes. Mad is not induced in all differentiating cell types, suggesting that other, possibly tissue-restricted, proteins might act in similar switch mechanisms to effect changes in transcriptional programs. We have also developed an approach to identification of the gene targets for Myc:Max complexes. By employing an immunoisolation procedure, we have begun characterization of several clones whose expression levels correlate with those of c-myc. Further identification of Myc-regulated genes may allow us to determine the molecular mechanism by which Myc governs cell proliferation and differentiation.

Mnt: A Novel Max-interacting Protein and Myc Antagonist

We have identified a novel Max-binding protein, Mnt, which belongs to neither the Myc nor the Mad families (Hurlin et al. 1997). Mnt interacts with Max in vivo and functions as a transcriptional repressor of reporter genes containing promoter-proximal CACGTG sites. Mnt:Max complexes also efficiently suppress Myc-dependent activation from the same promoter. Transcription repression by Mnt maps to a 13 amino acid N-terminal region related to the Sin3 interaction domain (SID) of Mad proteins. This region of Mnt mediates interaction with mSin3 corepressor proteins and its deletion converts Mnt from a repressor to an activator and from a suppressor of Myc-dependent transformation to a cooperating oncogene. This latter result suggests that Mnt and Myc regulate an overlapping set of target genes in vivo. Expression of mnt RNA is observed in many tissues and in both proliferating and differentiating cells. Likewise, Mnt protein is expressed in many proliferating cell types in culture where both Myc:Max and Mnt:Max complexes are detected. An exception is P19 embryonal carcinoma cells, where Mnt is expressed and in a complex with Max, but Myc proteins are not detected. Mnt is likely to be a key regulator of Myc activities in vivo and, in addition, may possess Myc-independent functions.